Thrombolytic agents with modified kringle domains

ABSTRACT

This invention relates to analogs of thrombolytic agents having a modified kringle domain. More specifically, this invention is directed to TPA type compounds wherein modifications occur in the kringle 1 and kringle 2 domains. This invention is also directed to analogs with one or more changes in the kringle domain of urokinase-type compounds. The compounds of this invention are pharmaceutically useful having particular use in the same manner as TPA and urokinase.

FIELD OF INVENTION

This invention is to analogs of thrombolytic agents having a modifiedkringle domain. More specifically this invention is directed to TPA typecompounds wherein modifications in the kringle 1 and kringle 2 domainsoccur. This invention is also directed to analogs with one or morechanges in the kringle domain of urokinase-type compounds. The compoundsof this invention are pharmaceutically useful having particular use inthe same manner as TPA and urokinase.

BACKGROUND OF INVENTION

Plasminogen activators (PA's) are known to play a central role in thefibrinolytic process. Their mechanism of action involves conversion ofthe inactive proenzyme plasminogen to plasmin, an active serineprotease, which in turn degrades the fibrin network of a clot. There aretwo known types of physiologically relevant PA's: urokinase-typeplasminogen activators (uPA) and tissue-type plasminogen activators(TPA). TPA's are found to be far more specific in their action thanuPA's showing a higher affinity for fibrin than uPA's and a specificityfor the blood clot itself.

Tissue plasminogen activator (TPA) is a glycosylated 66,000 daltonsserum protease of 527 amino acid residues produced by the vascularendothelial cells. It is a single polypeptide chain made up of fivemajor domains: the fibronectin finger domain (F_(t)), the growth factordomain (G_(t)), the kringle 1 (K_(t) 1) and kringle 2 (K_(t) 2) domainsand the active site (A_(t)). It is also known that TPA becomes activatedupon binding to fibrin. When digested with plasmin, the one-chain TPA iscleaved at a single site (Arg 275-Ile 276) converting it to a two-chainform, a light and a heavy chain connected by a disulfide bridge. Thelight chain derived from the carboxy terminus contains the catalyticsite. It has a great deal of homology with other serine proteases anddoes not bind fibrin. The heavy chain derived from the amino terminus isa multi-domain entity with homology to several other serine proteins. Itcontains a finger domain, a growth factor domain and two kringles. Thekringles are triple disulfide structures. The heavy chain has been foundto bind fibrin. TPA is cleared by the liver through the heavy chain.

Our recent results have shown that fibrin binding of TPA is localized intwo domains. The finger domain contains a high affinity fibrin bindingsite and the kringle 2 domain a low affinity fibrin binding site. Theaffinity of the latter site is increased by plasminogen. Stimulation byfibrin and fibrinogen fragments however is mainly localized in thekringle 2 domain. Results obtained by another group (van Zonneveld andPannekoek, CLB, Amsterdam) suggest stimulation by fibrin does not occurwhen K1 is directly coupled to the light chain. These results lead tothe conclusion that the position of kringle 2 (adjacent to the lightchain) and its structure are essential for its role in fibrinstimulation of activity. When we compare the amino acid sequences of TPAK2 with TPA K1, the uPA kringle, and the five plasminogen kringles thereare only a limited number of amino acids that are unique for TPA K2.Particularly a group of amino acids around number 250 in the inner loopof the kringle is interesting in this respect.

Urokinase was first isolated from urine. It was then isolated fromcultured cells, e.g., kidney cell lines and recently by expression ofcDNA in E. coli or mammalian cells in culture. When urokinase isexpressed in E. coli, it must be renatured.

Urokinase is a single polypeptide of 411 amino acid residues with amolecular weight of 55,000 daltons. This single chain form, alsodesignated pro-urokinase (pro-u-PA) or single chain urokinase (scu-PA)has low activity, but it can be converted to a two-chain (tuc-PA) formby e.g., plasmin. The two chains stay connected to each other by asingle disulfide bridge. In u-PA, three domains can be discerned - agrowth factor (G_(u)), a kringle domain (Ku) and a protease domain(P_(u)).

The G_(u) domain can interact with a cellular receptor. The P_(u) domaincontains the active site residues serine, histidine, and asparaginewhich are usually found in serine proteases. The function of the k_(u)domain is unknown.

Homologous kringle domains also occur in plasminogen, factor XII,prothrombin and lipoprotein. In both t-PA and plasminogen, one or morekringle domains are involved in fibrin binding. This has not beenobserved in u-PA. Like t-PA, u-PA catalyses the conversion of theinactive proenzyme plasminogen to the active protease plasminogen. Inthe case of t-PA this conversion is greatly enhanced by fibrin, whereasfor u-PA this is not the case. Nevertheless, fibrin specificity for u-PAhas been observed. This observation has been explained by assuming thatu-PA preferentially activates fibrin-bound plasminogen.

Since the fibrin specificity of t-PA and u-PA is based on a differentmechanism, it should be possible in principle to combine the desiredproperties of both molecules in a single hybrid molecule. Such hybridshave been constructed, containing long pieces of u-PA and the fibrinbinding domains of t-PA or plasminogen. The results are disappointing.Although some fibrin affinity is present in some hybirds, they do notcompare to that of t-PA itself.

What is needed are novel t-PA and u-PA analogs which have a greateraffinity for fibrin clots and a longer half-life.

INFORMATION DISCLOSURE

Patent application PCT WO 87/03906 published Jul. 2, 1987 describes TPAanalogs comprising an active site, i.e., the protease domain, and one ormore domains selected from the finger domain, the growth factor domain,the kringle 1 domain, and the kringle 2 domain wherein the domainregions have been altered from their native arrangement with respect totheir order, occurrence, or both, provided that the overall molecularweight of the protein does not exceed 90,000 daltons and no domainappears more than twice. The compounds of the present invention aredifferent from the compounds described in WO 87/03906 in that thekringle_(t) 1 and kringle_(t) 2 domains are modified and have adifferent amino acid sequence from that found in TPA. In addition, PCTWO 87/03906 does not teach urokinase analogs.

SUMMARY OF THE INVENTION

The present invention provides thrombolytic agents with one or moremodified kringle domains.

The present invention further provides oligonucleotides having thefollowing nucleotide sequence:

P106: SEQ I.D. No: 1

P107: SEQ I.D. No: 2

P108: SEQ I.D. No: 3

P109: SEQ I.D. No: 4

P110: SEQ I.D. No: 5

P111: SEQ I.D. No: 6

P112: SEQ I.D. No: 7

P114: SEQ I.D. No: 8

P115: SEQ I.D. No: 9

P116: SEQ I.D. No: 10

P117: SEQ I.D. No: 11

P118: SEQ I.D. No: 12

P120: SEQ I.D. No: 13

P121: SEQ I.D. No: 14

P122: SEQ I.D. No: 15

P123: SEQ I.D. No: 16

The present invention further provides TPA analog compounds wherein thekringle_(t) domains are modified as follows: in kringle_(t) 1 the aminoacids at positions 160 to 165 are replaced by the amino acids found atpositions 248 to 253 of kringle_(t) 2; in kringle_(t) 2 the amino acidsat positions 248 to 253 are replaced by the amino acids found atpositions 160 to 165 of kringle_(t) 1. More specifically kringle_(t) 1is modified by substituting asparagine (N), arginine (R), arginine (R),leucine (L), threonine (T), tryptophan (W) respectively at positions 160to 165. More specifically kringle_(t) 2 is modified by substitutingalanine (A), glycine (G), lysine (K), tyrosine (Y), serine (S), serine(S) respectively at positions 248 to 253. Thus the amino acids whichnormally occur at positions 160 to 165 in kringle_(t) 1, i.e., A, G, K,Y, S, S are replaced by N, R, R, L, T, W, and the amino acids whichnormally occur at positions 248 to 253 of kringle_(t) 2, i.e., N, R, R,L, T, W are replaced by A, G, K, Y, S, S. Hereinafter kringle_(t) 1 andkringle_(t) 2 modified as described above will be referred to as k_(t) 1and k_(t) 2 respectively to distinguish these modified kringle_(t)domains from the naturally occurring domains K_(t) 1 and K_(t) 2.

Another embodiment of the present invention relates to TPA analogcompounds comprising an active or protease site (A) and one or moredomains selected from the finger domain (F), the growth factor domain(G_(t)), the modified kringle 1 domain (k_(t) 1), and the modifiedkringle 2 domain (k_(t) 2) wherein the domain regions have been alteredfrom the native arrangement with respect to their order, occurrence orboth provided that no domain appears more than twice. Native arrangementmeans the relationship of the domains identified for native or naturallyoccurring TPA which is known to be FGK1K2A.

The present invention also provides a human u-PA analog that is a betterthrombolytic agent than native u-PA in that it exhibits increased fibrinbinding and/or better stimulation by fibrin or combination of otherproperties. The present invention focuses on the kringle region of u-PAwherein the kringle_(u) is modified by replacing the amino acid residuesat position 118-123 inclusive of u-PA with the amino acids found atposition 248-253 inclusive of K_(t) 2 of t-PA. More specifically, K_(u)is modified by replacing glycine (G), Leucine (L), Lysine (K), Proline(P), Leucine (L) and Valine (V) with Asparagine (N), Arginine (R),Arginine (R), Leucine (L), Threonine (T) and Thyrptophan (W),respectfully.

The present invention describes the development of a human TPA analogthat is a better thrombolytic agent than native TPA in that it exhibitsone or more of the following properties: (1) a longer half-life thannative TPA; (2) enhanced affinity for fibrin clots; and (3) lower or nobinding to endogenous inhibitors, such as plasminogen activatorinhibitor #1.

The present invention focuses on both of t-PA's kringle structures whichhave relatively conserved amino acid sequences. Only K_(t) 2 appears tobe vital to TPA's effectiveness for fibrin enhanced activity. Thelongest stretched of non-conserved sequence between kringle_(t) 1 andkringle_(t) 2 is six amino acids. It is thought that this stretch mightbe responsible for fibrin binding affinity of K_(t) 2 versus K_(t) 1.Therefore, this region is switched from kringle_(t) 1 to kringle_(t) 2and vice versa. To accomplish this, both kringle_(t) structures must bereassembled from oligonucleotides containing the desired changes.

The modified kringle_(t) structures are enzymatically assembled fromoligonucleotide fragments that are chemically synthesized using asolid-phase phosphoramidite triester coupling approach. After enzymaticassembly of the oligonucleotide fragments, each kringle_(t) structure isindividually cloned and successfully sequenced using the dideoxysequencing methodology.

The key starting materials for making the compounds (to be used inmaking modified kringles_(t)) of the present invention are described inpatent application PCT WO 87/03906 published Jul. 2, 1987, which isincorporated herein by reference.

In the present invention, small changes in the K_(u) domain of u-PA aredescribed which improve the fibrin binding properties, but do not alterthe structure of the molecule very much. A u-PA molecule like this ispossibly better for thrombolytic therapy than either naturally occurringt-PA or u-PA and may lead to a lower dosage (and cheaper treatment) withless side effects.

DETAILED DESCRIPTION OF INVENTION

As used herein, the domains of t-PA are denoted by subscript t. Thedomains of u-PA are denoted by subscript u.

As indicated hereinabove key starting materials for the preparation ofthe compounds of the present invention are described in patentapplication PCT WO 87/03906. These key starting materials are twoprototype genes having unique endonuclease restriction sites between thedomains of TPA designated herein F_(t), G_(t), K_(t) 1, K_(t) 2, andA_(t) for the finger, growth factor, kringle 1, kringle 2, and proteaseor active site domains respectively. It should be noted that the kringle1 (K_(t) 1) and kringle 2 (K_(t) 2) domains in the two prototype geneshave the nucleotide sequence which would give the natural amino acidsequence as found in TPA. These two prototype genes are deposited inaccordance with the Budapest Treaty. The host microorganisms are E. colistrains containing plasmids designated pTPA-B1,2,3,4(a) andpTPA-B1,2,3,4 and illustrated in Charts 1 and 2 respectively. Thedeposits were made with the Northern Regional Research Center, Peoria,Ill., USA. pTPA-B1,2,3,4(a) was deposited on Aug. 29, 1986 and assignedAccession Number NRRL B-18106. pTPA-B1,2,3,4 was deposited on Nov. 28,1986 and assigned Accession Number NRRL B-18142.

Starting materials for the preparation of the compounds of the presentinvention are the DNA moieties for the modified kringle_(t) structureswhich are enzymatically assembled from oligonucleotide fragments thatwere chemically synthesized using a solid-phase phosphoramidite triestercoupling approach. After enzymatic assembly of the oligonucleotidefragments, each kringle_(t) structure was individually cloned andsequenced using the dideoxy sequencing methodology. The DNA sequence ofthe modified kringle 1 (k_(t) 1) domain (block 3') is depicted in Chart3 wherein the differences between the DNA sequences of the modifiedkringle_(t) 1 and the natural kringle_(t) 1 are noted with underlining.The DNA sequence of the modified kringle 2 domain (k_(t) 2) (block 4')is depicted in Chart 4. In addition to modifying the DNA coding for theamino acids at positions 248 to 253 in kringle_(t) 2 long stretches ofguanosines (G) and cytidines (C) were eliminated. Each nucleotide changeis marked with an asterisk (*). The dashed underlining portion of thesequence represents a linker at the 3'-end of the sequence for cloningpurposes.

The materials and enzymes used in preparing the modified kringletdomains are obtained as follows. All the reagents for oligonucleotidesynthesis are ordered from Applied BioSystems (ABI) except foracetonitrile and methanol (HPLC grade) which are both from Burdick andJackson. Acetonitrile is dried by refluxing over calcium hydridefollowed by distillation. Acrylamide, bis-acrylamide, bromophenol blue,xylene cyanol and TEMED (tetramethylenediamine) are all purchased fromBioRad. Urea is from Bethesda Research Laboratories. Lysozyme, flucoseand ampicillin are obtained from Sigma. T4 polynucleotide kinase and T4DNA ligase, as well as all the restriction enzymes are purchased fromNew England BioLabs. RNase A is from Pharmacia and Reverse Transcriptasefrom Seikagaku America, Inc. The dNTP's and ddNTP's are purchased fromPharmacia. γ-^(32p) ATP and α-^(32p) dATP are ordered from Amersham. TheE. coli AG1 competent cells come from Stratagene. The tryptone and yeastextract for Lennox Broth both are purchased from Difco Labs as in theantibiotic medium 2 for the agar plates. The nitrocellulose filterscomes from Schleicher and Schuell.

All new oligonucleotides (P106, P107, P108-P112, P114-P118, P120-P123)required for the assembly of the modified kringle 1 (k_(t) 1) andkringle 2 (k_(t) 2) are synthesized on an ABI 380B DNA synthesizer usingthe solid-phase phosphoramidite triester coupling approach (S. L.Beaucage, M. H. Caruthers, Tetrahedron Letters 22:1859-1862, 1981). Theoligonucleotides are cleaved off the support and partially deprotectedusing the synthesizer's deprotection program followed by treatment withconcentrated ammonium hydroxide at 50° C. overnight. They are purifiedby polyacrylamide gel electrophoresis under denaturing conditions aspreviously described in detail (New York Theriault, et al., Nucleosidesand Nucleotides 5:15-32, 1986). The oligonucleotides are sized bylabeling with γ-^(32p) ATP in the presence of T4 polynucleotide kinaseand running a polyacrylamide gel under denaturing conditions againstknown standards.

The k_(t) 1 DNA (block 3') is made using the same oligonucleotides asused for kringle_(t) 1 DNA described in PCT WO 87/03906 exceptoligonucleotides P32 and P42 are replaced by P106 and P107 respectivelyin order to change the six amino acids as described above. The modifiedk_(t) 1 block 3' is made up of a total of 20 oligonucleotides varying inlength from a 21-mer to a 33-mer and has the structures set forth inChart 4. The k_(t) 2 DNA (block 4') is made using a total of 12oligonucleotides varying in length from a 41-mer to a 50-mer. Thestructures of the oligonucleotides are depicted in Chart 4.Oligonucleotides P24 to P31, P33 to P41 and P43 are purchased from NewEngland BioLabs. All the other oligonucleotides P106, P107, P108-P112,P114-P118, P120-P123 ranging in length from a 30-mer to a 50-mer aresynthesized in very good yield (99.0%+per coupling).

The oligonucleotides are deprotected and purified by polyacrylamide gelelectrophoresis using the conditions published by New York Theriault, etal., ibid. They are desalted on Waters SepPak C-18 cartridges. Theirsizes and purities are confirmed by labeling with γ-^(32p) ATP andpolynucleotide kinase and running them on a polyacrylamide gel againstknown standards.

The k_(t) 1 (block 3') is assembled following the strategy elaborated inChart 5. The fragments are divided in two groups and ligated to yieldI121 J125 and K148 L146. After purification and isolation, these areannealed and ligated to yield block 3'. After purification bypolyacrylamide gel electrophoresis (10%) under non-denaturing conditionsand isolation, block 3' is successfully cloned into pBR322, andsequenced by dideoxy methodology (E. Y. Chen, P. H. Seeburg, DNA4:165-170, 1985; A. M. Maxam, W. Gilbert, Methods in Enzymol.,65:499-560, 1980).

The enzymatic assembly of k_(t) 2 (block 4') is carried out asillustrated in Chart 6 and yielded M-182 N-185 and O-133 P-130. Afterpurification and isolation these are annealed and ligated to yield block4'. After purification by polyacrylamide gel electrophoresis (10%) undernon-denaturing conditions and isolation, block 4' is cloned into pBR322and sequenced as described above.

In cloning the modified kringle_(t) 1 and modified kringle_(t) 2 DNA theplasmids pBR322/Bam H1 and C1a 1, and pBR322/Bam H1 and Hind III areprepared. The circular pBR322 is cut with the appropriate restrictionenzymes following the conditions outlined by the supplier for theenzymes being used. Purification is then done on a horizontalpreparative 1% agarose gel in 1×E buffer (0.04M Tris acetate, 0.002MEDA) at 30 V overnight. The DNA was electroeluted from the agarose bandin 0.5×E buffer at 50 V for 4 hours, phenol extracted, and ethanolprecipitated.

After enzymatic assembly of the oligonucleotides to generate the desiredsequence, each of the kringle_(t) structures is ligated into itsrespective linear plasmid using T4 DNA ligase, 100 ng of plasmid DNA,and approximately 100-fold molar excess of insert DNA. The ligation isdone in 20 μl of ligation buffer (0.5M Tris-HCl, pH 7.4; 0.1M MgCL₂ ;0.1M DTT; 10 mM ATP) at 15° C. overnight. Equimolar amounts (0.2 nmol)of the gene fragments are labeled, annealed, ligated and the resultingligated products purified as described previously in detail (New YorkTheriault, et al., ibid., and Biotechniques 6:470-474 (1988); Jay, etal., J. Biol. Chem. 259:6311-6317, ™(1984).

E. coli AG1 competent cells from Strategene are used for transformationof the plasmid/insert ligation reaction mixture. Transformations aredone of each ligation reaction mixture, using 5 ng of reaction mixtureDNA for each transformation. The transformations are done on ice for 30minutes, heat shocked at 42° C. for 45 seconds, and then grown inL-broth at 37° C. for an hour before plating on agar plates containingampicillin. Details of the procedure can be found in Maniatis, et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,1982, and in Stratagene's product information.

All positive transformants are identified by hybridization experimentswith oligos used in the construction of the kringle_(t) structures. Theoligos chosen for hybridization are less than 25 bases long and have abase composition of approximately 50% G/C. The hybridizations and filterwashes are done at different temperatures depending on the length of theoligonucleotides being used. Modified kringle_(t) 1 is done 12 hours at42° C. and washed with 0.1×SSC at 50° C. for 30 minutes and at 55° C.for 30 minutes with fresh SSC. The oligo being used is P25 which is a 24mer and has 63% G/C content. Modified kringle_(t) 2 is done at 42° C.for 72 hours with P48, a 23 mer with 43% G/C content. Washings are donethe same way as for modified kringle_(t) 1. It is finally washed with0.2×SSC once at 42° C. Details of these protocols can be found inManiatis, et al., ibid. Positive transformants are identified byautoradiography.

To make sure that the identified positive transformants contained thecorrect sequence without any mutations, at least one clone each of themodified kringle 1 (k_(t) 1) and modified kringle 2 (k_(t) 2) iscompletely sequenced by the dideoxy sequencing procedure (R. B. Wallace,et al., Gene 16:21-26, 1981; E. Y. Chen, P. H. Seeburg, ibid.) Thepositives from autoradiography are grown up overnight at 37° C. in 5 mlof L-broth containing ampicillin. Plasmid DNA is isolated from theseovernight cultures by the alkaline lysis procedure. Details can be foundin Maniatis, et al., ibid. From each of these overnight cultures enoughDNA (about 250 ng) is isolated to run three sets of sequencingreactions. The DNA from the mini-preps is treated with DNase-freepancreatic RNase A (using 10 μg in 40 μl total volume) for one hour at37° C. The RNase is removed by phenol extraction and the DNA was ethanolprecipitated. The DNA is denatured with 2N NaOH/2 mM EDTA for 10 to 20minutes at room temperature. Priming with the sequencing templates isdone during ethanol precipitation. Dideoxy sequencing is done withreverse transcriptase following standard procedures for sequencing ofdouble-stranded DNA. Primers for sequencing are ones that are availablefrom New England BioLabs and/or oligonucleotides that were used in theassembly of blocks 3' and 4'.

The kringles of t-PA (K_(t) 1, K_(t) 2) are very similar to that of u-PA(K_(u)). Therefore, analogous changes as made in t-PA K_(t) 1 and givingit fibrin-binding properties are made in u-PA K. Specifically, theresidues numbered 118 to 123 in u-PA, glycine, leucine, lysine, proline,leucine, valine (GLKPLV) are replaced by the residues in the analogousposition of t-PA K2 numbered 248 to 253-asparagine, arginine, arginine,leucine, threonine, and tryptophan (NRRLTW). Such changes can beaccomplished in different ways, but the alteration of a DNA coding foru-PA is the most convenient method. The altered protein can be producedby expression of this altered DNA in a bacterial or mammalian expressionsystem. The DNA can be changed using oligonucleotide directedmutagenesis with DNA coding for u-PA as a staring material. cDNA codingfor u-PA has been described. A fragment of u-PA cDNA coding for at leastthat part of the kringle_(u) to be mutated is cut from the cDNA, usingone or more restriction endonucleases and isolated with agarose gelelectrophoresis and cloned in a commercially available cloning vector.The resulting plasmid is used for mutagenesis using published protocolsor commercially available kits. Alternatively, a complete synthetic cDNAcoding for the altered u-PA is constructed. Such construction requiresoligonucleotides synthesized according to available protocols. Anadvantage of the latter method is that the DNA sequence can differ fromthe natural one and comprise one or more unique restriction enzymerecognition sequences greatly facilitating further alterations

A. Preparation of t-PA analogs

The specific examples set forth below teach the preparation of plasmidscontaining cDNA encoding illustrative TPA analogs containing modifiedkringle 1 (k_(t) 1) or modified kringle 2 (k_(t) 2). Conventions used torepresent plasmids and fragments in Charts 1-7 are meant to bysynonymous with conventional representations of plasmids and theirfragments. Unlike the conventional circular figures, the single linefigures on the charts represent both circular and linear double-strandedDNA. With respect to the orientation of the resin sequence only theinitiation of transcription is depicted as occurring from left to right(5' to 3'). Asterisks (*) represent the bridging of nucleotides tocomplete the circular form of the plasmids. Fragments do not haveasterisks because they are linear pieces of double-stranded DNA.Endonuclease restriction sites are indicated above the line. Genemarkers are indicated below the line.

The various symbols used in Charts 1-7 have the following meanings inrespect to t-PA:

F=finger domain

G=growth factor domain

K1=Kringle 1 domain

K2=Kringle 2 domain

N=noncoding 3' sequence

k1=Modified Kringle 1 domain

k2=Modified Kringle 2 domain

A=active site

L=TPA leader sequence

T=5' region of TPAcDNA

P=middle region of TPAcDNA

a=polyadenylation signal sequence

AmpR=ampicillin resistance

Ori=pBR322 origin of replication

SV40=simian virus origin of replication

dhfr=mouse dihydrofolate reductase marker

CMV=cytomegalovirus promoter

EXAMPLE 1 Construction of FGk1A plasmid

Digest plasmid pTPA-B1, 2, 3, 4 (see Chart 2) partially with Hpa I andMst I and isolate the 4 kb fragment. This digest deletes the K2 region.The 4 kb fragment is then ligated together to generate the FGK1Aplasmid. The FGK1A plasmid is digested with C1a I and Bam HI whichyields a 3.8 kb fragment with the K1 region deleted. The deleted K1region can now be replaced with the C1a/Bam HI k1 fragment by ligatingit to the 3.8 kb fragment (with the K1 region deleted) therebygenerating the analog FGk1A.

EXAMPLE 2 Construction of FGk2A plasmid

The plasmid pTPA-B1, 2, 3, 4 is digested with EcoRV and Xma III. The 4kb fragment is isolated and ligated to the 230 bp k2 fragment obtainedby digesting the plasmid containing block 4' (pBR322 Bam HI/Hind III)with Hpa I and Xma III. The resulting plasmid contains the FGk2Aconstruct.

EXAMPLE 3 Construction of FGk1K2A plasmid

The plasmid pTPA-B1, 2, 3, 4 is digested with C1a I and Bam HI and the 4kb fragment is isolated. The plasmid containing block 3' is digestedwith C1a I and Bam HI and the 270 bp fragment containing the k_(t) 1 isisolated. The 4 kb C1aI/Bam HI is then ligated to the 270 bp C1aI/Bam HIfragment to yield the plasmid containing FGk1K2A.

EXAMPLE 4 Construction of pTPA-IE-FGk1A

The construction of pTPA0IE-FGk1A is a two step process. Step oneinvolves the creation of pTPA-FGk1A which contains the desired TPAanalog, the polyadenylation signal sequence from BGH and the selectablemarkers and replicons of pSVCOW and Step 2 involves the insertion of theCMV I.E. promoter. While the example below describes the insertion ofTPA analog FGk1A, other analogs (e.g., FGk1K2A or FGk2A) can be insertedusing the same enzymes and procedures (see Chart 7).

Plasmid pTPA-CDNA (described in Chart 21, page 77 of PCT/US86/02684) iscut with Bam HI and Bg1 II to yield a 120 base pair fragment which isgel isolated. Plasmid pSVCOW7 (described in Chart 20, page 76 ofPCT/US86/02684) is cut with Eco RI and Pvu II to yield a 600 base pairfragment containing the polyadenylation sequence of bovine growthhormone which is gel isolated. The TPA analog sequence is obtained bycutting plasmid pFGk1A (Example 1 above) with Bg1 II and Ba1 I to obtaina 1.7 kilobase fragment which is gel isolated. A second sample ofpSVCOW7 is cut with Eco RI and Bam HI to yield a 5.8 kilobase fragmentcontaining the markers and replicons of pSVCOW7 which are gel isolated.The four isolated fragments are ligated using T4 ligase to yield plasmidpTPA-FGk1A (8.2 kb) which is then cut with Bam HI, and the CMV-IEpromoter (760 bp) is inserted to form plasmid pTOA-IE-FGk1A. The CMV-IEpromoter is obtained from a Pst I and Sau 3A digestion of the human CMVgenome.

EXAMPLE 5 Transfection and Culturing of CHO cells

Plasmid pTPA-IE-FGk1A is transfected into Chinese hamster ovary (CHO)cells deficient in dihydrofolate reductase (dhfr) using the calciumphosphate method for transfection of DNA into cells which is describedin detail by Graham, et al. (in Introduction of Macromolecules intoViable Mammalian Cells, Alan R. Liss, Inc., New York, 1980, pp. 3-25).The cell line used is the mutant DXB-11 originally available from L.Chasin of Columbia University and completely described in Proc. Natl.Acad. Sci. USA 77:4216-4220 (1980). The above method for transfectionrelies on the fact that cells which incorporate the transfected plasmidsare no longer dhfr deficient and will grow in Dulbecco's modifiedEagle's medium plus proline.

From the cells transfected with pTPA-IE-FGk1A, clones are isolated,which, when grown in a monolayer for two days, synthesize at least 10 ngFGk1A per million cells. From cells with pIETPA-FGk1A-dhfr clones areisolated which synthesize at least 100 ng TPA per million cells.Expression of the TPA analog can be detected by radioimmunoassay,enzymatic assay or Western blot techniques.

The following Table 1 demonstrates the production of the TPA analogs byCHO cells. The following Table 2 shows that by replacing the 6 aminoacids in Kringle_(t) 1 with the 6 amino acids from Kringle_(t) 2, wehave given k_(t) 1 properties similar to K_(t) 2, i.e., FGk1A activityis now stimulated in the presence of fibrin.

                  TABLE 1                                                         ______________________________________                                        Analog           I.U./ml*                                                     ______________________________________                                        Control medium    0.4                                                         FGk1A            92.3                                                         FGk1K2A          112.9                                                        ______________________________________                                         *Determined by using a chromogenic assay including plasminogen, the           synthetic substrate S2251, and CNBrdigested fibrinogen.                  

                  TABLE 2                                                         ______________________________________                                               ΔA405/min.sup.2 -M (×10.sup.3)                                      -Fibrinogen  +Fibrinogen                                                                              Enhancement                                  Analog   Fragments    Fragments  Factor                                       ______________________________________                                        FGK1A    29.2           228       7.8                                         FGk1A    18.5         2,180      117.8                                        ______________________________________                                    

B. Preparation of u-PA analogs

The specific example set forth below teach the preparation of plasmidscontaining cDNA encoding illustrative u-PA analogs containing themodified kringle_(u).

EXAMPLE 6

1. cDNA is prepared from polyA+RNA isolated from HT1080 and polymerasechain-reaction (PCR) technique with SEQ ID NO: 17 (oligonucleotide#1_(u)) and SEQ ID NO: 18 (oligonucleotide #2_(u)). These primer. Fromthis cDNA, u-PA coding cDNA is amplified with the polymerasechain-reaction (PCR) technique with5'-AGCAGATCGGAGACCGCAG-3'(oligonucleotide #1_(u)) and5'-TGGCCAGAGGGGTCTGGGCA-3' (oligonucleotide #2_(u)). Theseoligonucleotides are derived from the published sequence of u-PA cDNA.

2. Commercially available linkers containing a HindIII recognitionsequence are ligated to the amplified cDNA for u-PA. The resultingfragment is cut with HindIII and inserted in HindIII digested pUCplasmid. The resulting plasmid pUC u-PA is used for site directedmutagensis.

3. An expression plasmid is constructed from pUC u-PA (PUC is availablefrom Pharmacia) and a peV2 t-PA, a t-PA expression plasmid previouslydescribed by J. H. Verheijen et al, EMBO Journal, Vol, 5, pp. 3525-3530(1986). The plasmid pUC u-PA is cut with HindIII and StuI and the smallfragment containing the u-PA coding sequence is isolated. Plasmid peV2pre-t-PA, a derivative of peV2 t-PA (Verheijen et al), containing a SV40promoter followed by the t-PA signal sequence and the rabbit β-globinpoly-A signal, but missing most of the t-PA coding sequence, is cut withBgIII, made blunt ended with nuclease, and cut with HindIII. The largefragment is isolated and ligated with the HindIII-StuI fragment from pUCu-PA. The resulting plasmid p2V2 u-PA contains the SV40 promoterfollowed by the u-PA coding sequence and the rabbit β-globin poly-Asignal.

4. Mutagensis is performed in the pUC u-PA using the protocol ofMarotti, K. et al. Gene Analysis Techniques, 6:67-70 1989. One of thefollowing nucleotides, SEQ ID NO: 19 (oligonucleotide #3_(u)) or SEQ IDNO: 20 (oligonucleotide #4_(u)) is used for mutagensis.

4. Alternatively, the procedure can be performed in two steps using theprotocol of Marotti et al. First, the nucleotides coding for the sixamino acids (GLKPLV) originally present in u-PA are removed usingoligonucleotides SEQ ID NO: 21 (oligonucleotide #5_(u)) oroligonucleotide SEQ ID NO: 22 (oligonucleotide #6_(u)) followed by theinsertion of (NRRLTW) using oligonucleotides 3_(u) or 4_(u).

5. The presence of the mutation and the absence of any secondarymutations is confirmed by sequencing using a Boehringer Mannheim kit.

6. The plasmid is transfected into CHO cells and the cloned expressedurokinase analog is isolated according to the method of J. H. Verheijenet al, EMBO Journal, Vol. 5, pp. 3525-3530 (1986). ##STR1##

    __________________________________________________________________________    SEQUENCE LISTING                                                              __________________________________________________________________________    (1)                                                                              GENERAL INFORMATION:                                                       (i)                                                                              APPLICANT:               Nicole T. Hatzenbuhler; Keith R. Marotti;                                     Edward F. Rehberg; and Johan H. Verheijen         (ii)                                                                             TITLE OF INVENTION:      Thrombolytic Agents with Modified Kringle                                     Domains                                           (iii)                                                                            NUMBER OF SEQUENCES:     22                                                (iv)                                                                             CORRESPONDENCE ADDRESS:  Dvorak and Traub                                     (A) ADDRESSEE:           Livia Boyadjian, Esq.                                (B) STREET               20 Exchange Place, 37th Floor                        (C) CITY                 New York                                             (D) STATE                New York                                             (E) COUNTRY              United States                                        (F) ZIP                  10005                                             (v)                                                                              COMPUTER READABLE FORM:                                                       (A) MEDIUM TYPE:         Floppy Disc                                          (B) COMPUTER             IBM-PC Compatible                                    (C) OPERATING SYSTEM     MS-DOS, IBM-DOS                                      (D) SOFTWARE:            Wordperfect 5.1                                   (vi)                                                                             CURRENT APPLICATION DATA (if available):                                      (A) APPLICATION NUMBER:  07/689,079                                           (B) FILING DATE          June 7, 1991                                         (C) CLASSIFICATION                                                         (vii)                                                                            PRIOR APPLICATION DATA (if applicable)                                        (A) APPLICATION NUMBER:  PCT/US89/04247                                       (B) FILING DATE          October 4, 1989                                   (viii)                                                                           ATTORNEY/AGENT INFORMATION:                                                   (A) NAME: LIVIA BOYADJIAN                                                     (B) REGISTRATION NUMBER: (34,781)                                             (C) REFERENCE/DOCKET NUMBER:                                                                           2212.LB-6818                                      (ix)                                                                             TELECOMMUNICATION INFORMATION:                                                (A) TELEPHONE:           (212) 968-1300                                       (B) TELEFAX:             (212) 968-1307                                       (C) TELEX:               232843                                               (D) CABLE:               WORLDLEGIS NY                                     (2)                                                                           SEQ ID NO: 1                                                                  LENGTH :33                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        TACGTCTTTA AGAACCGCAG GCTGACGTGG GAG          33                              SEQ ID NO: 2                                                                  LENGTH :30                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        GCAGAACTCC CACGTCAGCC TGCGGTTCTT              30                              SEQ ID NO: 3                                                                  LENGTH :45                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        GATCCGTTAA CGACTGCTAC TTTGGCAATG GTTCAGCCTA CCGTG                                                                           45                              SEQ ID NO: 4                                                                  LENGTH :47                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        GCACGCACAG CCTCACCGAG TCAGGTGCCT CATGCCTTCC GTGGAAT                                                                         47                              SEQ ID NO: 5                                                                  LENGTH :49                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        TCCATGATCC TGATAGGCAA GGTTTACACA GCACAGAATC CTAGTGCTC                                                                       49                              SEQ ID NO: 6                                                                  LENGTH :41                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        AGGCACTGGG ACTCGGCAAA CATAATTACT GCCGGAATCC T 41                              SEQ ID NO: 7                                                                  LENGTH :46                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        GATGGCGATG CCAAGCCATG GTGCCACGTG CTGAAGGCAG GTAAGT                                                                          46                              SEQ ID NO: 8                                                                  LENGTH :47                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        GCGTGCCACG GTAGGCTGAA CCATTGCCAA AGTAGCAGTC GTTAACG                                                                         47                              SEQ ID NO: 9                                                                  LENGTH :47                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        CATGGAATTC CACGGAAGGC ATGAGGCACC TGACTCGGTG AGGCTGT                                                                         47                              SEQ ID NO: 10                                                                 LENGTH :50                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        AGTGCCTGAG CACTAGGATT CTGTGCTGTG TAAACCTTGC CTATCAGGAT                                                                      50                              SEQ ID NO: 11                                                                 LENGTH :41                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        CGCCATCAGG ATTCCGGCAG TAATTATGTT TGCCGAGTCC C 41                              SEQ ID NO: 12                                                                 LENGTH :46                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        GAGCTGTACT TACCTGCCTT CAGCACGTGG CACCATGGCT TGGCAT                                                                          46                              SEQ ID NO: 13                                                                 LENGTH :41                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        ACAGCTCAGA GTACTGTGAT GTGCCTTCCT GCGCAACCGC A 41                              SEQ ID NO: 14                                                                 LENGTH :46                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        TGCGGCCGGA GATACAGCCA GCCTCAGTTT CGCATCAAAG GAGGTA                                                                          46                              SEQ ID NO: 15                                                                 LENGTH :40                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        GCCGCATGCG GTTGCGCAGG AAGGCACATC ACAGTACTCT   40                              SEQ ID NO: 16                                                                 LENGTH :44                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        AGCTTACCTC CTTTGATGCG AAACTGAGGC TGGCTGTATC TCCG                                                                            44                              SEQ ID NO: 17                                                                 LENGTH :19                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        AGCAGATCGG AGACCGCAG                          19                              SEQ ID NO: 18                                                                 LENGTH :20                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        TGGCCAGAGG GGTCTGGGCA                         20                              SEQ ID NO: 19                                                                 LENGTH :45                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        TGGTGCTATG TGCAGGTGAA CCGCAGCCTG ACGTGGCAAG AGTGC                                                                           45                              SEQ ID NO: 20                                                                 LENGTH :45                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        GCACGCTTGC CACGTCAGGC TGCGGTTCAC CTGCACATAG CACCA                                                                           45                              SEQ ID NO: 21                                                                 LENGTH :36                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        TGGTGCTATG TGCAGGTGCA AGAGTGCATG GTGCAT       36                              SEQ ID NO: 22                                                                 LENGTH :36                                                                    TYPE :nucleotide                                                              STRANDEDNESS :single                                                          TOPOLOGY :linear                                                              SEQUENCE DESCRIPTION :                                                        ATGCACCATG CACTCTTGCA CCTGCACATA GCACCA       36                              __________________________________________________________________________

We claim:
 1. A tissue plasminogen activator (tPA) compound comprisingthe tPA active site (A_(t)) and a modified tPA kringle 1 domain(k_(t) 1) in which the amino acid sequence Ala Gly Lys Tyr Ser Ser atpositions 160-165 is replaced by the amino acid sequence Asn Arg Arg LeuThr Trp, and a combination of any or none of the tPA finger domain(F_(t)), the tPA growth factor domain (G_(t)), the natural tPA kringledomains (K_(t) 1, K_(t) 2), and a modified tPA kringle 2 domain (k_(t)2) in which the amino acid sequence Asn Arg Arg Leu Thr Trp at positions248-253 is replaced by the amino acid sequence Ala Gly Lys Tyr Ser Ser.2. The tissue plasminogen activator compound of claim 1, selected fromthe group consisting of F_(t) k_(t) 1 A_(t),F_(t) G_(t) k_(t) 1 A_(t),k_(t) 1 k_(t) 2 A_(t), F_(t) k_(t) 1 k_(t) 2 A_(t), F_(t) G_(t) k_(t) 1K_(t) 2 A_(t), and G_(t) k_(t) 1 k_(t) 2 A_(t).
 3. The tissueplasminogen activator compound of claim 1, selected from the groupconsisting ofF_(t) G_(t) k_(t) 1 k_(t) 1 A_(t), F_(t) G_(t) k_(t) 2k_(t) 1 A_(t), F_(t) F_(t) k_(t) 1 k_(t) 2 A_(t), and F_(t) F_(t) G_(t)k_(t) 1 k_(t) 2 A_(t).
 4. The tissue plasminogen activator compound ofclaim 2, wherein the compound isF_(t) G_(t) k_(t) 1 K_(t) 2 A_(t). 5.The tissue plasminogen activator compound of claim 1, selected from thegroup consisting ofG_(t) k_(t) 1 K_(t) 2 A_(t), F_(t) k_(t) 1 K_(t) 2A_(t), and k_(t) 1 K_(t) 2 A_(t).
 6. A compound of any of claims 1-3,whose molecular weight is no more than 90 kD.